| The red swamp crayfish,Procambarus clarkii,belonging to Crustacea,Decapoda,Cambaridae,Procambarus,is an important freshwater economic crayfish in China.P.clarkii has become popular because of its unique flavours in recent years.Therefore,the farming scale and the production of P.clarkii have been greatly increased.However,problems also occurred,such as degeneration of germplasm of P.clarkii caused by inbreeding and environmental damage in ecologically sensitive areas and ecological reserve caused by proliferation of P.clarkii.Monosex culture could improve the production of P.clarkii and reduce the biological invasion risk caused by P.clarkii to some extent.Insulin-like androgenic gland hormone(IAG)was reported to control the sex differentiation and to maintain the male secondary sexual characteristics in decapods,and its application achieved the monosex culture of Macrobrachium rosenbergii.The study of IAG in P.clarkii could be a breakthrough for it’s monosex culture.In this study,we designed one siRNA(GsRNA)based on the sequence of IAG and injected them into P.clarkii to explore the effect of GsRNA on IAG of P.clarkii.Then,we analyzed the mRNA(in three gonadal tissues and two nervous tissues)and miRNA(in two gonadal tissues)expression profiles after GsRNA interference.Finally,GsiRNA was used for long term interference of juveniles.When these juveniles grew up,the changes of internal and external characteristics were observed.Our results analyze the molecular regulatory mechanism of IAG in transcriptional and post-transcriptional level and provide an evidence for the important role of IAG in the reproduction of P.clarkii,which would further enrich the information of P.clarkii reproduction and lay a foundation for the sex manipulation of P.clarkii.The main research contents and results are as follows:(1)Analysis of mRNA expression profiles in gonadal and nervous tissues after siRNA interference.After injecting GsRNA into adult male P.clarkii(20 g-35 g),the expression level of IAG in androgenic gland(AG)was significantly inhibited,indicating that GsRNA could effectively inhibited the expression of IAG.The gonadal and nervous tissues that were collected from P.clarkii interfered with the siRNA were used for mRNA sequencing.Differential expression analysis indicated that the number of differentially expressed genes in nervous tissues was far more than that in gonadal tissues and the expression level of some genes encoding neuropeptide were found to changed after GsRNA interference,such as crustacean hyperglycemia hormone(CHH),corazonin,red pigment concentrating hormone;GO enrichment analysis showed that the differentially expressed genes in Brain(Br)and abdominal nerve cord(AN)were enriched in GO terms that related to protein biosynthetic and secretion and that related to reproduction;KEGG enrichment analysis showed that differentially expressed genes in Br and AN were significantly enriched in KEGG pathways that related to protein biosynthetic and reproduction.These results implied that nervous tissues might be involved in the regulation of IAG by secreting neuropeptide.Additionally,after silencing IAG by GsRNA,many ovary-related genes were up-regulated and many testis-related genes were down-regulated.The expression of many sex determination related genes also changed when GsRNA was injected into the P.clarkii.Differentially expressed genes in gonadal tissues were enriched in KEGG pathways that related to reproduction,indicating that IAG may play an important role in male sex regulation of P.clarkii.(2)Analysis of miRNA expression profiles in gonadal tissues after siRNA interference.The gonadal tissues used for mRNA sequencing were also used for miRNA sequencing to konw more about the regulation of IAG at the post-transcriptional level.Seven miRNAs with high expression level were identified from AG and testis(Te)in control group,including miR-1,miR-100,miR-279 a,SEQ14455＿4438,SEQ52799＿9422,SEQ23317＿6170 and SEQ52799＿9421.Five miRNAs related to sexual regulation were found to significantly differentially expressed after GsRNA interference and have high expression level in gonadal tissues,which were miR-263 a,miR-263 b,miR-2779,miR-133 and miR-34.The target genes of the differentially expressed miRNAs were predicted,and four sex-related genes were identified among the differentially expressed target genes,including testis-expressed protein 264-like,glycogen debranching enzyme,nanos and proliferating cell nuclear antigen.KEGG enrichment analysis of differentially expressed target genes showed that the differentially expressed target genes in GsRNA interference group were enriched in some KEGG pathways related to reproduction,such as MAPK signaling pathway,DNA replication and ovarian steroidogenesis(3)Long-term GsRNA interference and tracking of siRNATo know more about the significant role of IAG in reproduction of P.clarkii,GsRNA was chosen for long-term interference of juvenile P.clarkii(0.01 g-0.012 g).The result indicated that the first pair of abdominal appendages in the male juvenile P.clarkii developed abnormally after GsRNA interference,and the development of the male external sexual characteristics(i.e.,decrease in the degree of thickening and cuticularization of the first and second pleopods,degeneration of reverse spines on the third and fourth pereopods)in the male adult P.clarkii(8 g-15 g)of GsRNA interference group were delayed. |
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